In the prior art various solutions are known for analyzing the quantity of particles present in a fluid. In particular, flow cytometry consists of analyzing the optical or physical signals emitted by a particle intersecting the light beam of a laser or of an arc lamp in order to deduce there from the information on characterization and counting of the particles contained in the fluid.
Fluorescence analysis is generally carried out by means of the introduction and fixing of a large number of fluorescent markers, also referred to as fluorochromes, on different compounds of the solution. A specific excitation wavelength and therefore, potentially, an associated monochromatic light source corresponds to each fluorochrome.
Nevertheless, increasing the number of these markers is limited by the spectral overlap of the emission thereof. This is because overlap occurs when the fluorescence emission spectrum of a compound covers part of the emission spectrum of another compound.
To avoid this, several techniques can be used. The one used most often is the compensation method, which consists of reducing the fluorescence signal, or exciting signal, by the proportion of spectral overlap between two fluorochromes. Nevertheless, increasing the number of fluorochromes introduces significant complexity with regard to the analysis and consequently gives rise to a significant error rate on diagnosis.
One solution for overcoming these problems consists of introducing, on each excitation signal dedicated to a fluorochrome, a frequency modulation making it possible to identify the fluorescence emission by a frequency analysis in addition to chromatic analysis. For this purpose, an acousto-optical or electro-optical modulator can be used. This principle of multi-parameterized cytometric analysis has been the subject of several patent applications.
In particular U.S. Pre-Grant Publication No. 2003/0205682 is known, which describes the modulation of each wavelength at a given frequency. In addition, International Patent Application Publication No. WO 2006/111641 and U.S. Pre-Grant Publication No. 2004/0251436 disclose the use of acousto-optical modulators making it possible to select the wavelengths in a polychromatic light and to modify each of them specifically at a given frequency.
Frequency modulation, by means of acousto-optical or electro-optical modulators, gives rise to difficulty with regard to both the modulators and the light sources to be used. This is because the modulators have different characteristics according to the wavelength of use and must therefore be corrected for chromatic aberrations. In addition, the light source must have a sufficiently high emission frequency to obtain several analysis signals when the source signal is chopped. If the emission frequency is too low, the chopping of the source signal is limited and does not therefore make it possible to characterize sufficient markers.
Moreover, spectro-frequency modulation does not always make it possible to obtain reliable and satisfactory results, in particular when the number of fluorochromes is too high.
Finally, the use of acousto-optical modulators and laser sources having high emission frequencies represents high costs.